Prothrombin, Factor X, Factor IX, and Factor VII are synthesized in the liver initially as precursor molecules which then undergo several post translational modifications including vitamin K dependent formation of gamma-carboxyglutamic acid. In order to gain insight into the intrahepatic events that occur during the synthesis of the vitamin K-dependent blood coagulation proteins, the precursor forms of prothrombin from normal bovine liver will be purified and systematically characterized. An immunochemical approach will be used to isolate the precursor forms of prothrombin. Antibodies specific for abnormal prothrombin which do not crossreact with prothrombin will be purified from anti-abnormal prothrombin antisera using sequential immunoabsorption with agarose covalently linked to des-gamma-carboxy-fragment 1 and agarose covalently linked to prothrombin. Antibodies specific for prothrombin which do not crossreact with abnormal prothrombin will be isolated from anti-prothrombin antisera by absorption to an agarose column to which prothrombin is covalently bound and elution with EDTA. Noncarboxylated precursor forms of prothrombin will then be purified from a microsomal extract of normal bovine liver by affinity chromatography using agarose to which antibodies specific for abnormal prothrombin have been coupled. Carboxylated precursor forms of prothrombin will be purified by affinity chromatography using agarose to which antibodies specific for prothrombin have been coupled. Once purified the precursor forms of prothrombin and abnormal prothrombin will be characterized with regard to a) amino acid content including analysis for gamma-carboxyglutamic acid, b) the sequence of the NH2-terminal fifth of each precursor, c) carbohydrate content for hexoses, hexosamines and sialic acid, d)proteolysis products in the presence of Factor Xa and thrombin, e) molecular weight and f) isoelectric point.